The influenza A virus can undergo transcription and replication entirely inside the nucleus. The newly synthesized viral ribonucleoproteins (vRNPs) are later exported for incorporation into virions at the plasma membrane (Nayak et al., 2004). The viral proteins NS1 and NS2, designated as non-structural (NS) proteins in early studies (Lamb and Choppin, 1983), are both encoded by the smallest influenza virus segment, segment 8 (Inglis et al., 1979; Lamb et al., 1980).
NS1 has multiple functions during virus infection (Krug et al., 2003), including the inhibition of the early interferon-α/β-independent (IFN-α/β) antiviral response of cells by blocking the posttranscriptional processing of cellular antiviral pre-mRNAs (Fortes et al., 1994; Kim et al., 2008). The C-terminal domain of NS1, named the effector domain, binds the 30 kDa subunit of the cleavage and polyadenylation specificity factor (CPSF) (Meveroff et al., 1998) and the poly(A)-binding protein II (PABII) via residues 223-237 (Chen et al., 1999). These binding events prevent processing of the 3′ ends of cellular pre-mRNAs and thus their nuclear export. The C-terminal domain also directly targets the export machinery and the nuclear pore complex (Satterly et al., 2007). NS1 also blocks the cellular antiviral response mediated by protein kinase R (PKR) (Bergmann et al., 2000; Hatada et al., 1999; Lu et al., 1997) by direct binding to that protein (Tan and Katze, 1998). It also enhances the translation of the viral mRNAs (de la Luna et al., 1995; Enami et al., 1994; Park and Katze, 1995), and shuts off cellular protein synthesis, by interacting with the eukaryotic translational factor 4GI (eIF4GI) (Aragon et al., 2000; Burgui et al., 2003) and possibly with the host mRNA binding protein guanine-rich sequence factor 1 (GRSF-1) (Park et al., 1999).
Relative to NS1, the NS2 protein is much less well characterized. Although NS2 was initially designated as a non-structural protein (Lamb et al., 1980), it has been shown to exist in purified viral particles (Richardson and Akkina, 1991). NS2 also accumulates preferentially in the nuclei of infected eukaryotic cells (Greenspan et al., 1985; Smith et al., 1987). Some studies have suggested a role for NS2 in regulating viral RNA replication (Bullido et al., 2001; Odagiri et al., 1990; Odagiri et al., 1994), thereby providing a possible explanation for its nuclear accumulation. The function of NS2 during the viral cycle became more apparent when O'Neill and colleagues (O'Neill et al., 1998) showed that NS2 was the adaptor between the cellular nuclear export machinery Crm1 and the newly amplified viral genomic segments (vRNPs). They then renamed NS2 nuclear export protein (NEP).
The central role of NS2/NEP in vRNP nuclear export through Crm1 was later confirmed by in vivo (Iwatsuki-Norimoto et al., 2004; Neumann et al., 2006) and in vitro (Akarsu et al., 2003) studies. Conflicting data, however, have also been published, where the viral matrix protein M1 was sufficient for the nuclear export of vRNPs in the absence of NS2/NEP (Bui et al., 2000) and the viral nucleoprotein NP interacted directly with Crm1 (Elton et al., 2001). Nevertheless, the studies showing the interaction of NS2/NEP with Crm1 (Akarsu et al., 2003; Neumann et al., 2000; O'Neill et al., 1995) and with M1 (Akarsu et al., 2003; Yasuda et al., 1993) and of M1 with the vRNPs (Baudin et al., 2001; Bui et al., 2000) suggest the following model for the nuclear export of the newly synthesized vRNPs: the C-terminal segment of M1 binds to NP, which associates with the vRNA, and the nuclear localization signal (NLS) in the N-terminus terminus of M1 binds to the C-terminal region of NS2/NEP. In turn, the N-terminal region of NS2/NEP recognizes Crm1and permits the nuclear export of the vRNPs (Boulo et al., 2007).